Finian J. Leeper

The biosynthesis and regulation of bacterial prodiginines

The red-pigmented prodiginines are bioactive secondary metabolites produced by both Gram-negative and Gram-positive bacteria. Recently, these tripyrrole molecules have received renewed attention owing to reported immunosuppressive and anticancer properties. The enzymes involved in the biosynthetic pathways for the production of two of these molecules, prodigiosin and undecylprodigiosin, are now known. However, the biochemistry of some of the reactions is still poorly understood. The physiology and regulation of prodiginine production in Serratia and Streptomyces are now well understood, although the biological role of these pigments in the producer organisms remains unclear. However, research into the biology of pigment production will stimulate interest in the bioengineering of strains to synthesize useful prodiginine derivatives.

Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2‐methyl‐3‐n‐amyl‐pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces

The biosynthetic pathway of the red‐pigmented antibiotic, prodigiosin, produced by Serratia sp. is known to involve separate pathways for the production of the monopyrrole, 2‐methyl‐3‐n‐amyl‐pyrrole (MAP) and the bipyrrole, 4‐methoxy‐2,2′‐bipyrrole‐5‐carbaldehyde (MBC) which are then coupled in the final condensation step. We have previously reported the cloning, sequencing and heterologous expression of the pig cluster responsible for prodigiosin biosynthesis in two Serratia sp. In this article we report the creation of in‐frame deletions or insertions in every biosynthetic gene in the cluster from Serratia sp. ATCC 39006. The biosynthetic intermediates accumulating in each mutant have been analysed by LC‐MS, cross‐feeding and genetic complementation studies. Based on these results we assign specific roles in the biosynthesis of MBC to the following Pig proteins: PigI, PigG, PigA, PigJ, PigH, PigM, PigF and PigN. We report a novel pathway for the biosynthesis of MAP, involving PigD, PigE and PigB. We also report a new chemical synthesis of MAP and one of its precursors, 3‐acetyloctanal. Finally, we identify the condensing enzyme as PigC. We reassess the existing literature and discuss the significance of the results for the biosynthesis of undecylprodigiosin by the Red cluster in Streptomyces coelicolor A3(2).

Anticancer and immunosuppressive properties of bacterial prodiginines

Bacterial prodiginines are a family of red-pigmented, tripyrrolic compounds that display numerous biological activities, including antibacterial, antifungal, antiprotozoal, antimalarial, immunosuppressive and anticancer properties. Recently, significant progress has been made in understanding the biosynthesis and regulation of bacterial prodiginines. An understanding of the biosynthesis of prodiginines will allow engineering of bacterial strains capable of synthesizing novel prodiginines through rational design and mutasynthesis experiments. Bacterial prodiginines and synthetic derivatives are effective proapoptotic agents with multiple cellular targets, and they are active against numerous cancer cell lines, including multidrug-resistant cells, with little or no toxicity towards normal cell lines. A synthetic derivative, GX15-070 (Obatoclax), developed through structure-activity relationship studies of the pyrrolic ring A of GX15, is in multiple Phase I and II clinical trials in both single and dual-agent studies to treat different types of cancer. Therefore, prodiginines have real therapeutic potential in the clinic.

Structure, mechanism and catalytic duality of thiamine-dependent enzymes

Abstract.Thiamine is an essential cofactor that is required for processes of general metabolism amongst all organisms, and it is likely to have played a role in the earliest stages of the evolution of life. Here, we review from a structural perspective the enzymatic mechanisms that involve this cofactor. We explore asymmetry within homodimeric thiamine diphosphate (ThDP)-dependent enzyme structures and discuss how this may be correlated with the kinetic properties of half-of-the-sites reactivity, and negative cooperativity. It is likely these structural and kinetic hallmarks may arise through reciprocal coupling of active sites. This mode of communication between distant active sites is not unique to ThDP-dependent enzymes, but is widespread in other classes of oligomeric enzyme. Thus, it appears likely to be a general phenomenon reflecting a powerful mechanism of accelerating the rate of a chemical pathway. Finally, we speculate on the early evolutionary history of the cofactor and its ancient association with protein and RNA.

SYNTHESIS OF AND ASYMMETRIC INDUCTION BY CHIRAL POLYCYCLIC THIAZOLIUM SALTS

Abstract Three different chiral bicyclic thiazolium salts, 10, 16 and 19 , have been synthesised in enantiomerically pure form. These salts all catalysed the formation of benzoin and butyroin from benzaldehyde and butyraldehyde respectively and the product had the R -configuration in each case with e.e.s in the range 10–33%. Possible reasons for these results are discussed.

Imaging sialylated tumor cell glycans in vivo

Cell surface glycans are involved in numerous physiological processes that involve cell‐cell interactions and migration, including lymphocyte trafficking and cancer metastasis. We have used a bioorthogonal metabolic labeling strategy to detect cell surface glycans and demonstrate, for the first time, fluorescence and radionuclide imaging of sialylated glycans in a murine tumor model in vivo. Peracetylated azido‐labeled N‐acetyl‐man‐nosamine, injected intraperitoneally, was used as the metabolic precursor for the biosynthesis of 5‐azidoneuraminic, or azidosialic acid. Azidosialic acid‐labeled cell surface glycans were then reacted, by Staudinger ligation, with a biotinylated phosphine injected intraperitoneally, and the biotin was detected by subsequent intravenous injection of a fluorescent or radiolabeled avidin derivative. At 24 h after administration of NeutrAvidin, labeled with either a far‐red fluorophore or 111In, there was a significant azido‐labeled N‐acetyl‐mannosamine‐dependent increase in tumor‐to‐tissue contrast, which was detected using optical imaging or single‐photon‐emission computed tomography (SPECT), respectively. The technique has the potential to translate to the clinic, where, given the prognostic relevance of altered sialic acid expression in cancer, it could be used to monitor disease progression.—Neves, A. A., Stöckmann, H., Harmston, R. R., Pryor, H. J., Alam, I. S., Ireland‐Zecchini, H., Lewis, D. Y., Lyons, S. K., Leeper, F. J., Brindle, K. M. Imaging sialylated tumor cell glycans in vivo. FASEB J. 25, 2528–2537 (2011). www.fasebj.org

Imaging cell surface glycosylation in vivo using "double click" chemistry.

Dynamic alterations in cell surface glycosylation occur in numerous biological processes that involve cell–cell communication and cell migration. We report here imaging of cell surface glycosylation in live mice using double click chemistry. Cell surface glycans were metabolically labeled using peracetylated azido-labeled N-acetylgalactosamine and then reacted, in the first click reaction, with either a cyclooctyne, in a Huisgen [3 + 2] cycloaddition, or with a Staudinger phosphine, via Staudinger ligation. The second click reaction was a [4 + 2] inverse electron demand Diels–Alder reaction between a trans-cyclooctene and a tetrazine, where the latter reagent had been fluorescently labeled with a far-red fluorophore. After administration of the fluorescent tetrazine, the bifunctional cyclooctyne-cyclooctene produced significant azido sugar-dependent fluorescence labeling of tumor, kidney, liver, spleen, and small intestine in vivo, where the kidney and tumor could be imaged noninvasively in the live mouse.

Development and evaluation of new cyclooctynes for cell surface glycan imaging in cancer cells

Two reagents have been synthesized for selective labeling of cell surface azidoglycans, an unusually stable version of a dibenzocyclooctyne (TMDIBO) and a third-generation difluorinated cyclooctyne (DIFO3). Both syntheses are efficient with minimal purification, and the dibenzocyclooctyne is stable under basic and acidic conditions. Flow cytometric measurements with azidosugar labeled cancer cells, in which these reagents were linked to the fluorophore Alexa Fluor 647, gave a signal-to-background ratio of up to 35 with TMDIBO as compared to ≈10 for DIFO3 and ≈5 for a phosphine reagent. TMDIBO-based probes should have applications in molecular imaging of cell surface glycans in vivo.

Biosynthesis of Vitamin B12

Vitamin B12 (cyanocobalamin) is the normal isolated form of coenzyme B12 (adenosylcobalamin), a structure of marvellous architecture and amazing biological activity. It belongs to the family of tetrapyrroles which includes inter alia the haems and the chlorophylls. This review begins with a brief overview of the biosynthesis of tetrapyrroles in general but then concentrates on recent research on B12 biosynthesis. The first main section reviews the biosynthesis of uro’gen III, the last common precursor of all natural tetrapyrroles, concentrating particularly on the three enzymes, porphobilinogen synthase, hydroxymethylbilane synthase and uro’gen III synthase. Crystal structures are available for the second of these enzymes and a new proposal is presented for its detailed mode of action. The second main section reviews the recent discovery of the complete biosynthetic pathway from uro’gen III to hydrogen-obyrinic acid in Pseudomonas denitrificans, which has revealed some beautiful and totally unexpected chemistry. A short section then describes the many similarities and some differences in the chemistry used by the micro-aerophilic organism Propionibacterium shermanii for the synthesis of coenzyme B12 compared with that seen in the aerobic Ps. denitrificans. Finally an account is given of the remarkable steps needed to complete the synthesis of the coenzyme in both organisms.

Bacterial Biosynthetic Gene Clusters Encoding the Anti-cancer Haterumalide Class of Molecules BIOGENESIS OF THE BROAD SPECTRUM ANTIFUNGAL AND ANTI-OOMYCETE COMPOUND, OOCYDIN A

Background: Oocydin A is an anticancer haterumalide with strong antimicrobial activity against agriculturally important plant pathogenic fungi and oomycetes. Results: The oocydin A gene cluster has been identified and characterized in four plant-associated enterobacteria. Conclusion: The ooc gene cluster is organized in three transcriptional units encoding enzymes that belong to a growing class of trans-acyltransferase polyketide synthases. Significance: Oocydin A has potential agricultural, pharmacological, and chemotherapeutic applications. Haterumalides are halogenated macrolides with strong antitumor properties, making them attractive targets for chemical synthesis. Unfortunately, current synthetic routes to these molecules are inefficient. The potent haterumalide, oocydin A, was previously identified from two plant-associated bacteria through its high bioactivity against plant pathogenic fungi and oomycetes. In this study, we describe oocydin A (ooc) biosynthetic gene clusters identified by genome sequencing, comparative genomics, and chemical analysis in four plant-associated enterobacteria of the Serratia and Dickeya genera. Disruption of the ooc gene cluster abolished oocydin A production and bioactivity against fungi and oomycetes. The ooc gene clusters span between 77 and 80 kb and encode five multimodular polyketide synthase (PKS) proteins, a hydroxymethylglutaryl-CoA synthase cassette and three flavin-dependent tailoring enzymes. The presence of two free-standing acyltransferase proteins classifies the oocydin A gene cluster within the growing family of trans-AT PKSs. The amino acid sequences and organization of the PKS domains are consistent with the chemical predictions and functional peculiarities associated with trans-acyltransferase PKS. Based on extensive in silico analysis of the gene cluster, we propose a biosynthetic model for the production of oocydin A and, by extension, for other members of the haterumalide family of halogenated macrolides exhibiting anti-cancer, anti-fungal, and other interesting biological properties.